| Objective: Based on our research group’s previous studies on Hei Dihuang Wan,the intervention effect of Hei Dihuang Wan on CRF rats was discussed by establishing a 5/6 nephresectoid animal model rat,and the possible mechanism of inhibiting renal cell apoptosis of Hei Dihuang Wan was discussed based on IGF-1/PI3K/AKT signaling pathway,so as to provide experimental data support for the clinical application of Hei Dihuang Wan.Methods: After 50 rats were fed adaptively for 1 w,10 rats were randomly selected as blank group,and the remaining rats were reproduced by 5/6nephrectomy.After 8w of modeling operation,the serum of the model was detected,and the successfully constructed rats were randomly divided into model group,Hei Dihuang Wan group and Hei Dihuang Wan +JB1 group.Administration group was given the corresponding group name drug intervention,blank group and model group were given the same amount of normal saline,once a day for consecutive 8w.The body weight and general behavior of the rats were observed and recorded.The rats were killed after gavage and specimens were collected.Serum creatinine and urea nitrogen were detected.HE staining was used to examine the pathological tissue of kidney.Masson staining: The collagen deposition area of renal interstitium was observed.TUNEL method was used to detect the apoptosis rate of renal cells in each group.Serum IGF-1in each group was determined by Elisa.Western blot method was used to detect the protein levels of IGF-1R,PI3 K,AKT,p-Akt,Bax and Bcl-2 in renal tissues of each group.Immunohistochemistry: Phosphorylated Akt(p-Akt),Akt and PI3 K protein were detected in kidney tissue.Results:(1)Hei Dihuang Wan could improve the general condition of rats and significantly increase the body weight(P<0.05).(2)Scr and BUN levels in model group were significantly higher than those in other groups,with statistical significance(P<0.05),and the effect of reducing Scr and BUN in Hei Dihuang Wan group was better than that in Hei Dihuang Wan +JB1 group(P<0.05).(3)HE staining showed that the renal tissue structure was clear and no pathological changes were observed in the blank group.In other groups,there were glomerular cystic enlargement,mesangial cell and matrix hyperplasia,renal tubule vacuolar degeneration,basement membrane thickening,a large number of inflammatory cell infiltration and collagen accumulation in renal interstitium,and renal fibrosis gradually aggravated.The above conditions were improved in administration group,especially in Hei Dihuang Pill group.(4)Masson staining showed that compared with blank group,the deposition area of blue collagen fibers in renal interstitium of model group was significantly increased(P<0.01),and all administration groups were reduced compared with model group,and Hei Dihuang Pill group had the best improvement(P<0.05).(5)TUNEL fluorescence staining showed that the apoptosis rate of kidney cells in blank group was the lowest,and that in model group was significantly increased(P<0.01).Compared with model group,the apoptosis rate of kidney cells in all groups after administration was decreased,especially in Hei Dihuang Wan group(P<0.05).(6)ELISA showed that compared with blank group,serum IGF-1 level in model group was significantly decreased(P<0.01);Compared with model group,serum IGF-1 level in all administration groups was increased,especially in Hei Dihuang Pill group(P<0.05).(7)Western blot results showed that compared with blank group,the apoptosis-related protein BAX was significantly up-regulated,BCL-2 was down-regulated,and the expressions of the signaling path-related proteins IGF-1R,PI3 K and P-Akt were decreased in model group(P<0.05).Compared with model group,the expressions of BCL-2,IGF-1R,PI3 K and P-Akt in Hei Dihuang Pill group were increased,while the level of BAX protein was decreased in each administration group,with statistical significance(P<0.05).After the addition of IGF-1R blocker JB1,the above changes were lower than those in Hei Dihuang Wan group,and the AKT protein expression in all groups had no significant changes(P> 0.05).(8)Immunohistochemical results showed that compared with blank group,the expressions of PI3 K and P-Akt in kidney tissue of model group were decrease(P<0.05).Compared with model group,the expressions of PI3 K and P-Akt in kidney tissue of Hei Dihuang Wan group were significantly up-regulated(P < 0.01).Compared with Hei Dihuang Wan group,the expressions of PI3 K and P-Akt in kidney tissue of rats in Hei Dihuang Wan+JB1 group were lower than those in Hei Dihuang Wan group(P < 0.05),but there was no significant difference in the expression of AKT among all groups(P >0.05).Conclusion: 1.Hei Dihuang Wan can obviously improve the general condition of CRF model rats,protect renal function and renal pathological changes.2.Hei Dihuang Wan can reduce the apoptosis rate of kidney cells in CRF model rats,up-regulate BCL-2 protein,and down-regulate BAX protein,which can effectively inhibit the apoptosis of kidney cells.3.Hei Dihuang Wan inhibits renal cell apoptosis by regulating IGF-1/PI3K/AKT signaling pathway. |
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